Pharmacological inhibition of poly(ADP-ribose) polymerase inhibits angiogenesis

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which plays an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP are being considered as treatment for cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation, and were also reported to be protective against untoward effects exerted by certain anticancer drugs. Here we show that pharmacological inhibition of PARP with 3-aminobenzamide or PJ-34 dose-dependently reduces VEGF-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. These results suggest that treatment with PARP inhibitors may exert additional benefits in various cancers and retinopathies by decreasing angiogenesis.     

Differentiative pathway activated by 3-aminobenzamide, an inhibitor of PARP, in human osteosarcoma MG-63 cells

This study describes the molecular mechanism by which treatment with 3-AB, a potent inhibitor of PARP, allows human osteosarcoma MG-63 cells to restrict growth and enter differentiation. Our findings show that in MG-63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3-AB-treatment, the poly(ADP-ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, beta-catenin, c-Jun, c-Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N-cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming of gene expression and the activation of differentiation.     

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.     

Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation

Oxidative stress induced cell injury is reported to contribute to the pathogenesis of cerebral ischemia. Reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide radical along with nitric oxide and peroxynitrite generated during ischemia-reperfusion injury, causes the overactivation of poly (ADP-ribose) polymerase (PARP) leading to neuronal cell death. In the present study we have evaluated the effects of PARP inhibitor, 8-hydroxy-2 methyl-quinazolin-4-[3H]one (NU1025) in H2O2 and 3-morphilinosyndonimine (SIN-1) induced cytotoxicity in PC12 cells as well as in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Exposure of PC12 cells to H2O2 (0.4 mM) and SIN-1 (0.8 mM) resulted in a significant decrease in cell viability after 6 h. Pretreatment with NU1025 (0.2 mM) restored cell viability to approximately 73 and 82% in H2O2 and SIN-1 injured cells, respectively. In MCAO studies, NU1025 was administered at different time points (1 h before reperfusion, immediately before reperfusion, 3 h after reperfusion and 6 h after reperfusion). NU1025 at 1 and 3 mg/kg reduced total infarct volume to 25% and 45%, respectively, when administered 1 h before reperfusion. NU1025 also produced significant improvement in neurological deficits. Neuroprotection with NU1025 was associated with reduction in PAR accumulation, reversal of brain NAD depletion and reduction in DNA fragmentation. Results of this study demonstrate the neuroprotective activity of NU1025 and suggest its potential in cerebral ischemia.     

Cleavage of poly(ADP-ribose) polymerase during apoptosis induced by nicotinamide at high concentrations in tobacco suspension cells

Apoptosis induced by high concentrations of nicotinamide in tobacco suspension cells was observed. When cells were treated with 250 mM nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180–200 bp, condensation and peripheral distribution of nuclei chromatin and positive reaction to the TUNEL assay. In addition, the degradation of poly (ADP-ribose) polymerase (PARP) was also detected. This indicates that caspase-3-like activity is involved in apoptosis in cultured tobacco cells induced by high-concentration nicotinamide. However, as an inhibitor of PARP, nicotinamide has a contrary effect on apoptosis at low concentrations, which suggests that nicotinamide plays a dual role depending on to its concentration in cells.     

1-Oxo-3,4-dihydroisoquinoline-4-carboxamides as novel druglike inhibitors of poly(ADP-ribose) polymerase (PARP) with favourable ADME characteristics

A novel 3,4-dihydroisoquinol-1-one-4-carboxamide scaffold was designed as the basis for the development of novel inhibitors of poly(ADP-ribose) polymerase (PARP). Synthesis of 3,4-dihydroisoquinol-1-one-4-carboxylic acids was achieved using the previously developed protocol based on the modified Castagnoli-Cushman reaction of homophthalic anhydrides and 1,3,5-triazinanes as formaldimine synthetic equivalents. Employment of 2,4-dimethoxy groups on the nitrogen atom of the latter allowed preparation of 2,3-unsubatituted 3,4-dihydroquinolone core building blocks. Iterative synthesis and in vitro biological testing of the amides resulting from the amidation of these carboxylic acids allowed not only drawing important structure-activity generalisations (corroborated by in silico docking simulation) but also the identification of the lead compound, 4-([1,4''-bipiperidine]-1''-carbonyl)-7-fluoro-3,4-dihydroisoquinolin-1(2H)-one, as the candidate for further preclinical development. The lead compound as well as its des-fluoro analog were compared to the approved PARP1 inhibitor, anticancer drug Olaparib, in terms of their molecular characteristics defining druglikeness as well as experimentally determined ADME parameters. The newly developed series demonstrated clear advantages over Olaparib in terms of molecular weight, hydrophilicity, human liver microsomal and plasma stability as well as plasma protein binding. Further preclinical investigation of the lead compound is highly warranted.     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.     

多聚ADP-核糖聚合酶抑制剂对高浓度锌损伤 PC12细胞的保护作用

探讨多聚ADP-核糖聚合酶(PARP)抑制剂3-氨基苯甲酰胺(3-AB)对400μmo1/L氯化锌损伤PC12细胞的保护作用及其对锌造成的细胞死亡类型的影响.应用MTT法,免疫细胞化学和Western印迹分别测定PC12细胞的存活率和PARP活性;用Hoechst 33342/PI荧光双染色、膜联蛋白V结合实验及DNA断裂分析等方法检测细胞死亡类型.结果表明在400μmol/L氯化锌的作用下,细胞存活率降至(22.7±4.6)%,PARP活性增强,坏死、凋亡和正常细胞百分比分别为(58.4±6.3)%、(18.0±5.6)%及(23.6±4.2)%;3-AB使细胞存活率提高至(76.9±4.7)%,PARP活性减弱,坏死细胞百分数降至(19.2±5.2)%,而正常和凋亡细胞百分数增加到(43.3±1.9)%和(37.5±6.5)%.实验证明,PARP参与了高浓度锌诱导的PC12细胞损伤,抑制PARP活性可提高细胞的存活率,而这种保护作用在于减少细胞的坏死而非凋亡.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Effect of transient focal ischemia of mouse brain on energy state and NAD levels: no evidence that NAD depletion plays a major role in secondary disturbances of energy metabolism

It has been proposed that NAD depletion resulting from excessive activation of poly(ADP-ribose) polymerase is responsible for secondary energy failure after transient cerebral ischemia. However, this hypothesis has never been verified by measurement of ATP and NAD levels in the same tissue sample. In this study, we therefore investigated the effect of transient focal cerebral ischemia on the temporal profiles of changes in the levels of energy metabolites and NAD. Ischemia was induced in mice by occluding the left middle cerebral artery using the intraluminal filament technique. Animals were subjected to 1-h ischemia, followed by 0, 1, 3, 6, or 24 h of reperfusion. During ischemia, ATP levels, total adenylate pool, and adenylate energy charge dropped to approximately 20, 50, and 40% of control, respectively, whereas NAD levels remained close to control. Energy state recovered transiently, peaking at 3 h of recovery (ATP levels and total adenylate pool recovered to 78 and 81% of control). In animals subjected to reperfusion of varying duration, the extent of ATP depletion was clearly more pronounced than that of NAD. The results imply that depletion of NAD pools did not play a major role in secondary disturbances of energy-producing metabolism after transient focal cerebral ischemia. Changes in ATP levels were closely related to changes in total adenylate pool (p<0.001). The high energy charge after 6 h of reperfusion (0.90 versus a control value of 0.93) and the close relationship between the decline of ATP and total adenylate pool suggest that degradation or a washout of adenylates (owing to leaky membranes) rather than a mismatch between energy production and consumption is the main causative factor contributing to the secondary energy failure observed after prolonged recovery.     

The inhibition of poly(ADP-ribose) polymerase enhances growth rates of ataxia telangiectasia cells

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which is activated in response to genotoxic insults by binding damaged DNA and attaching polymers of ADP-ribose to nuclear proteins at the expense of its substrate NAD+. In persons affected with ataxia telangiectasia (A-T), associated mutations in the ataxia telangiectasia mutated gene render cells unable to cope with the genotoxic stresses from ionizing radiation and oxidative damage, thus resulting in a higher concentration of unrepaired DNA damage and the activation of PARP in an uncontrolled manner. In primary A-T fibroblasts, we observed a 58-96% increase in PARP activity and a concomitant loss of cellular NAD+ and ATP content. PARP protein by Western blot analysis increased only slightly in these cells, supporting the observation that the steady state levels of DNA damage is higher in A-T cells than in normals. When treated with PARP inhibitors 3-aminobenzamide or 1,5-dihydroisoquinoline, cellular growth rates reached those observed in normal fibroblast cultures. The improvement of cellular growth and NAD+ levels in A-T cells with PARP inhibition suggests that the cellular metabolic status of A-T cells is compromised and the inhibition of PARP may relieve some of the drain on cellular pyridine nucleotides and ATP. Thus, therapy utilizing PARP inhibitors may provide a benefit for individuals affected with A-T.     

Effects of poly(ADP-ribose) polymerase inhibition on dysfunction of non-adrenergic non-cholinergic neurotransmission in gastric fundus in diabetic rats

Diabetes mellitus compromises nitric oxide (NO)-mediated endothelium-dependent relaxation of blood vessels, which has been linked to the excessive generation of reactive oxygen species. There are also deleterious effect on nitrergic innervation, contributing to autonomic neuropathy symptoms such as impotence and gastroporesis. Poly(ADP-ribose) polymerase (PARP) is a nuclear protein stimulated by DNA damage, caused, for example, by oxidative stress. Activation has been linked to impaired endothelial nitric oxide synthase (eNOS)-mediated vasodilation in experimental diabetes. There is no information on the potential role of PARP in nitrergic nerve dysfunction, therefore, the aim was to examine the effects of PARP inhibition, using 3-aminobenzamide (3-AB) on neurally mediated gastric fundus relaxation in streptozotocin-induced diabetic rats. Eight weeks of diabetes caused a 42.5% deficit in maximum relaxation of in vitro gastric fundus strips to electrical stimulation of the non-adrenergic non-cholinergic innervation. This was largely prevented or corrected (4 weeks of treatment following 4 weeks of untreated diabetes) by 3-AB. Diabetes also markedly attenuated the maintenance of relaxation responses to prolonged stimulation, and this was partially corrected by 3-AB treatment. Experiments in the presence of the NOS inhibitor, N(G)-nitro-L-arginine, and/or blockade of the co-transmitter, vasoactive intestinal polypeptide, by alpha-chymotrypsin, showed that the beneficial effects of 3-AB were primarily due to improved nitrergic neurotransmission. Thus, PARP plays an important role in defective nitrergic neurotransmission in experimental diabetes, which may have therapeutic implications for treatment of aspects of diabetic autonomic neuropathy.     

Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-ribose)polymerase inhibitors

Structure based drug design of a series of novel 1,4-benzoxazin-3-one derived PARP-1 inhibitors are described. The synthesis, enzymatic & cellular activities and pharmacodynamic effects are described. Optimized analogs demonstrated inhibition of poly-ADP-ribosylation in SW620 tumor bearing nude mice through 24 h following a single dose.     

Three-component Castagnoli-Cushman reaction with ammonium acetate delivers 2-unsubstituted isoquinol-1-ones as potent inhibitors of poly(ADP-ribose) polymerase (PARP)

An earlier described three-component variant of the Castagnoli-Cushman reaction employing homophthalic anhydrides, carbonyl compound and ammonium acetate was applied towards the preparation of 1-oxo-3,4-dihydroisoquinoline-4-carboxamides with variable substituent in position 3. These compounds displayed inhibitory activity towards poly(ADP-ribose) polymerase (PARP), a clinically validated cancer target. The most potent compound (PARP1/2 IC₅₀ = 22/4.0 nM) displayed the highest selectivity towards PARP2 in the series (selectivity index = 5.5), more advantageous ADME prameters compared to the clinically used PARP inhibitor Olaparib.